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Chromatography
Introduction:
Chromatography is the general name given to the methods by which two or more
compounds in a mixture are physically separated by distributing between two phases: a stationary
phase which can be a solid or liquid supported on a solid and a mobile phase, either a gas or a liquid
which flows continuously around the stationary phase.
Chromatography is a method by which a mixture is separated by distributing its components
between two phases. The stationary phase remains fixed in place while the mobile phase carries
the components of the mixture through the medium being used. The stationary phase acts as a
constraint on many of the components in a mixture, slowing them down to move slower than the
mobile phase. The movement of the components in the mobile phase is controlled by the
significance of their interactions with the mobile and/or stationary phases. Because of the
differences in factors such as the solubility of certain components in the mobile phase and the
strength of their affinities for the stationary phase, some components will move faster than
others, thus facilitating the separation of the components within that mixture.
Discovery of chromatography technique:
Chromatography was first devised at the University of Kazan by the
Italian-born Russian scientist Mikhail Tswett in 1900. He developed the
technique and coined the term chromatography in the first decade of the
20th century, primarily for the separation of plant pigments such as
chlorophyll, carotenes, and xanthophylls.
Principle:
Chromatography is based on the principle where molecules in mixture applied onto the surface
or into the solid, and fluid stationary phase (stable phase) is separating from each other while
moving with the aid of a mobile phase. It is based on the difference in movement of individual
components of a mixture through the stationary phase under the influence of mobile phase. For
example, a mixture of red and blue ink can be separated by chromatography. A drop of the mixture is
placed on the chromatogram. The component of the ink, which is less
adsorbed on the chromatogram, moves with the mobile phase while the less adsorbed component
remains almost stationary.
Components of chromatography:
Chromatography Columns: Separation of sample components before detection is the essence of
chromatographic techniques. A chromatographic system uses a column to achieve the desired
separation. A column comprises of a tube packed with a stationary phase on which separation takes
place based on physico – chemical interactions of separating compounds with the stationary phase. The
mobile phase or the carrier gas elutes less weakly retained components first followed by more strongly
retained [Link] column dimensions and composition is based on the chromatographic
technique selected and the degree of required separation.
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Chromatographic Detectors: After separation the individual components reach the detector which
provides response in terms of the area or peak height depending on the amount of the eluting
compound. Each chromatographic separation technique offers a range of detectors depending on the
nature of eluting compounds. Detection can be specific for a particular compound or a range of
compounds or it can depend on some physical properties such as reflective index of the mobile phase.
Such detectors are referred to as bulk property detectors in comparison to selective or specific
detectors.
Chromatographic Data: Chromatographic separations appear as peaks in the chromatogram except
for thin-layer chromatography for which separate zones are seen on the plate. Each peak represents a
sample component and is characterized by its retention time under defined operating conditions. For
purposes of quantification the peak height or more accurately the area under the peak defines the
amount of the component present in the mixture.
Types of Chromatography:
1. Absorption Chromatography: In the process of adsorption
chromatography, different compounds are adsorbed on the adsorbent to
different degrees based on the absorptivity of the component. Here also,
a mobile phase is made to move over a stationary phase, thus carrying
the components with higher absorptivity to a lower distance than that
with lower absorptivity.
2. Thin Layer Chromatography
In the process of thin-layer chromatography (TLC), the mixture of
substances is separated into its components with the help of a glass
plate coated with a very thin layer of adsorbent, such as silica gel and
alumina. The plate used for this process is known as chrome plate.
The solution of the mixture to be separated is applied as a small spot
at a distance of 2 cm above one end of the plate. The plate is then
placed in a closed jar containing a fluid termed as an eluant, which
then rises up the plate carrying different components of the mixture
to different heights.
3. Column Chromatography
Column chromatograph is the technique used to separate the components of a mixture using a column
of suitable adsorbent packed in a glass tube, as shown in the figure below. The mixture is placed on the
top of the column, and an appropriate eluant is made to flow down the column slowly.
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Depending upon the degree of adsorption of the components on
the wall adsorbent column, the separation of the components
takes place. The component with the highest absorptivity is
retained at the top, while the other flow down to different
heights accordingly.
4. Partition chromatography:
In this process, a continuous differential partitioning of components of a mixture into a stationary phase
and mobile phase takes place. The example of partition chromatography can be seen in paper
chromatography. In this process, chromatography paper is used
as a stationary phase which is suspended in a mixture of
solvents that act as a mobile phase. Here, we put a spot at the
base of the chromatographic paper with the mixture to be
separated and as the solvent rises up this paper, the components
are carried to different degrees depending upon their retention
on the paper. The components are thus separated at different
heights.
Chromatograph: An analytical instrument that uses chromatography is called a
chromatograph. The combination of the instrument with a detector enables quantitative
analysis of substances and is commonly used in many fields.
Chromatogram: A chromatogram is the result obtained when analysis is performed using
a chromatograph (an analyzer). The horizontal axis of the chromatogram expresses time
and the vertical axis expresses signal value (intensity, absorbance, count, etc.). A
calibration curve is then used to convert the signal value to a concentration (amount) and
to derive the concentration of the substance.
HPLC: High-performance liquid chromatography (HPLC) is an analytical technique to
separate, identify, and quantify components in a mixture.
It is the main chromatography technique used in most
laboratories worldwide. High-performance liquid
chromatography or commonly known as HPLC is an
analytical technique used to separate, identify or quantify
each component in a mixture. The mixture is separated
using the basic principle of column chromatography and
then identified and quantified by [Link] the 1960s, the column chromatography LC with its
low-pressure suitable glass columns was further developed to the HPLC with its high-pressure adapted
metal columns. HPLC is thus basically a highly improved form of column liquid chromatography.
Instead of a solvent being allowed to drip through a column under gravity, it is forced through under
high pressures of up to 400 atmospheres.
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Applications of chromatography:
Pharmaceutical and Clinical Testing: Chromatography plays an important role in the safety of
pharmaceuticals. Pharmaceutical companies use chromatography to quantify and analyze compounds
for contaminants.
Food and Beverage: Quality control within the food and beverage industry can be enacted through
chromatography. In the food industry, chromatography is used to separate and analyze additives,
vitamins, proteins, amino acids, and other nutritional compounds in food items. Chromatography can
also be used to determine expiration dates by distinguishing the number of organic acids present as well
as to detect any harmful toxins that may have been added to the food item.
Environmental and Chemical Industry: The chemical industry must adhere to numerous
environmental safety precautions. Perfluoroalkyl substances, also known as PFAS, have become a
persistent threat to the human body and the environment.
By using solid-phase extraction, liquid chromatography, and mass spectrometry, we can detect
PFAS in the environment and our drinking water, even at very low limits.
Drug Testing: Chromatography can be very useful in drug testing and clinical toxicology reports.
Chromatography can separate and analyze substances found in urine samples. When running a clinical
toxicology report, drug testing a new employee or testing a professional athlete for performance-
enhancing drugs, chromatography determines what substances have been taken through an analysis of a
urine sample which ultimately determines if any harmful or illicit drugs have been used.
Security: Security practices are a unique industry where chromatography can be utilized. Gas
chromatography can be used to determine volatile gases, furthering safety measures at locations such as
airports and large gatherings with similar safety precautions like concerts and sporting events to
eliminate deadly threats.
Rf value in chromatography:
RF stands for retention factor in paper chromatography, or the distance a fluid compound moves up a
plate of chromatography. For each particular solvent, all compounds have a common RF value, and RF
values are used to equate unknown samples with known compounds.
Why water is not used in paper chromatography?
It is preferable to use a less polar solvent, such as ethanol, so that the non-polar compounds will travel
up the paper while the polar compounds will stick to the paper, separating them.
